The primary objective of this pilot project is to establish a small blood volume assay that can be used in[unreadable] pediatric SLE patients, a critically underserved population. We will establish and validate quantitative assays[unreadable] of apoptotic cell clearance and cytokine secretion in response to apoptotic cell phagocytosis using small[unreadable] blood volumes, by using flow cytometry to quantify clearance, and multiplex cytokine assays to quantify[unreadable] secretion of several cytokines in very small volumes of supernatant. These assays will then be applied in a[unreadable] pilot way to a population of pediatric patients with SLE.[unreadable] Apoptotic cells, a potentially important antigen in SLE, are normally cleared by phagocytes in an antiinflammatory,[unreadable] tolerance-inducing way. Dysregulation of this process is thought to lead to systemic[unreadable] autoimmunity. We propose that during active disease, monocytes from SLE patients have diminished antiinflammatory[unreadable] (TGF- beta and IL-10) and increased pro-inflammatory (TNF-alpha, IL-1, IFN-a, IL-12) cytokine[unreadable] secretion in response to apoptotic cells, which contributes to SLE propagation, disease flares, and tissue[unreadable] damage. The relevance of such pathways in pediatric SLE is unknown. We have shown that adult SLE[unreadable] patients have strikingly decreased anti-inflammatory cytokine secretion in response to apoptotic cells[unreadable] compared to normal controls. However, this defect in TGF-beta secretion did not reflect a defect in uptake,[unreadable] indicating that the SLE monocytes are capable of phagocytosing the apoptotic cells.[unreadable] The relatively large blood volumes required for these assays present a significant challenge to directly[unreadable] addressing these pathways in children. However, with the small blood volume assays and multiplex ELISA[unreadable] proposed in this pilot project, 16 cytokines can be assayed from a single 250 mu l supernatant sample (an 8-[unreadable] fold reduction in supernatant volume). Thus, we should be able to generate the necessary supernatant[unreadable] volume with only 2 cc of blood, making this assay accessible to the pediatric SLE population.[unreadable] These techniques may have broad applicability for Dr. Sule, as she begins to grow her career in pediatric[unreadable] rheumatology. They may also enable studies in the pediatric population with rheumatic diseases which are currently not pursued due to logistic issues and difficulties with scaling down sample volumes.